Glycated proteins and advanced glycation end products (AGE) accumulate slowly in vascular and renal tissues with age, and more rapidly in inflammatory disease states. AGE contribute to cellular damage, particularly, diabetic tissue injury, by at least two major mechanisms: modulation of cellular functions through interactions with specific cell surface receptors, and alteration of the extracellular matrix leading to the formation of protein cross-links. Studies suggest that glycated protein and AGE interactions with cells may promote inflammatory processes and oxidative cellular injury. Diseases where glycated protein and AGE accumulation is a suspected etiological factor include vascular complications of diabetes, ventricular hypertrophy, atherosclerosis, angiopathy, myocarditis, nephritis, arthritis, glomerulonephritis, microangiopathies, renal insufficiency and Alzheimer's disease.
The exact mechanisms by which high plasma glucose, as seen in diabetes, causes microvascular damage are not completely understood. One potential mechanism by which hyperglycemia can be linked to microangiopathies is through the process of non-enzymatic glycation of critical proteins (1-3). Non-enzymatic glycation, i.e. the linking of proteins with glucose, leads to the formation of glycated proteins. The first step in this glycation pathway involves the non-enzymatic condensation of glucose with free amino groups in the protein, primarily the epsilon-amino groups of lysine residues, forming the Amadori adducts. These early glycation products can undergo further reactions such as rearrangements, dehydration and condensations to form irreversible advanced glycation end products (AGE). AGE are a highly reactive group of molecules whose interaction with specific cell-surface receptors, are thought to lead to pathogenic outcomes. Accumulation of glycated proteins is found in the basement membrane of patients with diabetes and is thought to be involved in the development of diabetic nephropathy and retinopathy (4,5). Inhibitors of AGE formation, such as aminoguanidine, prevent development of diabetes complications, including diabetic retinopathy (6-8).
The best characterized AGE receptor is RAGE, receptor for AGE (3). Several in vitro and in vivo studies have shown that blocking RAGE either by antibodies or by adding a soluble form of the receptor inhibits diabetic vasculopathy including diabetic atherosclerosis (9-11). Apart from AGE, RAGE appears to mediate the binding of several other ligands that are involved in normal physiology as well as pathology (12,13). Thus, merely blocking RAGE might have other unintended consequences. Moreover, since blocking RAGE would lead to accumulation of AGE in circulation, the long-term effects of blocking RAGE are unknown and may be more harmful than the pathology sought to be prevented.
There are currently no efficient methods for determining compounds effective for the inhibition of AGE or glycated protein accumulation. What is needed are methods and compositions for detecting compounds that can be used to interfere with the production of glycated proteins or AGE. Such detection methods and compositions need to be high throughput and capable of determining effective compounds easily.